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. 2013 Sep;83(1-2):119-29.
doi: 10.1007/s11103-013-0030-7. Epub 2013 Mar 4.

Cysteine protease enhances plant-mediated bollworm RNA interference

Ying-Bo Mao  1 , Xue-Yi Xue, Xiao-Yuan Tao, Chang-Qing Yang, Ling-Jian Wang, Xiao-Ya Chen
Affiliations

Cysteine protease enhances plant-mediated bollworm RNA interference

Ying-Bo Mao et al. Plant Mol Biol. 2013 Sep.

Abstract

Oral ingestion of plant-expressed double stranded RNA (dsRNA) triggers target gene suppression in insect. An important step of this process is the transmission of dsRNA from plant to midgut cells. Insect peritrophic matrix (PM) presents a barrier that prevents large molecules from entering midgut cells. Here, we show that uptake of plant cysteine proteases, such as GhCP1 from cotton (Gossypium hirsutum) and AtCP2 from Arabidopsis, by cotton bollworm (Helicoverpa armigera) larvae resulted in attenuating the PM. When GhCP1 or AtCP2 pre-fed larvae were transferred to gossypol-containing diet, the bollworm accumulated higher content of gossypol in midgut. Larvae previously ingested GhCP1 or AtCP2 were more susceptible to infection by Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV), a dsRNA virus. Furthermore, the pre-fed larvae exhibited enhanced RNAi effects after ingestion of the dsRNA-expressing plant. The bollworm P450 gene CYP6AE14 is involved in the larval tolerance to gossypol; cotton plants producing dsRNA of CYP6AE14 (dsCYP6AE14) were more resistant to bollworm feeding (Mao et al. in Transgenic Res 20:665-673, 2011). We found that cotton plants harboring both 35S:dsCYP6AE14 and 35S:GhCP1 were better protected from bollworm than either of the single-transgene lines. Our results demonstrate that plant cysteine proteases, which have the activity of increasing PM permeability, can be used to improve the plant-mediated RNAi against herbivorous insects.

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Figures

Fig. 1
Fig. 1
Expression patterns of GhCP1 and AtCP2. a GhCP1 expression in cotton (G. hirsutum). Cotyledon of 1-week-old seedlings, and leaf (the second leaf from top), petal and ovule (day 3 post-anthesis) of adult plant (12 weeks old) were analyzed; b AtCP2 expression in leaf, stem, inflorescence and silique of 4-week-old plants of A. thaliana; Expression of GhCP1 (c) and AtCP2 (d) after wounding. The 4–week-old cotton plant and the Arabidopsis leaves were treated by wounding for indicated times. The expressions of GhCP1 in cotyledon and AtCP2 in leaf were set to 1. In wounding treatment, the expression level just before treatments was set to 1. Each investigation had at least three biological repeats, error bars represent standard deviation (SD)
Fig. 2
Fig. 2
Proteolytic activity of GhCP1 and AtCP2. a Sketch of GhCP1 and AtCP2 domains; b SDS-PAGE of purified proteins. His-tag fusion proteins of GFP, GhCP1 and AtCP2 were expressed in E. coli; c Proteolytic activity of purified His-GhCP1 and His-AtCP2. Purified proteins were added in reaction buffer and incubated at room temperature for indicated times as described in “Materials and methods”; d Effect of gut fluids on proteolytic activity of GhCP1 and AtCP2. Purified His-GhCP1 and His-AtCP2 proteins (final concentration: 2 mg/ml) were added to reaction buffer and incubated at room temperature for 1 h. +1 μl of gut fluid of 5th instar larvae was added to 100 μl reaction buffer; −no gut fluid added. Data are shown as mean ± SD
Fig. 3
Fig. 3
The effects of cysteine protease on bollworm midgut PM permeability to gossypol. Varied gossypol accumulation in midgut after bollworm ingestion of cysteine proteases. The 3rd instar larvae were fed with artificial diet supplemented with E. coli expressing His-tag fusion proteins of GFP, GhCP1 or AtCP2, respectively (a), or with the purified fusion proteins (b) for 8 h, and then transferred to gossypol (1 mg/g)-supplemented diet for another 16 h; gossypol content in midgut was measured by a phloroglucinol/HCl assay. To 10 g artificial diet, precipitates of 50 ml liquid culture of E. coli cells (OD 1.0), or purified His-GhCP1, His-AtCP2 or His-GFP proteins (10 mg each), respectively, were added; Accumulation of gossypol in midgut cells of the bollworms that were pre-fed with 35S:GhCP1 (c) or 35S:AtCP2 (d) plant leaves. The 3rd instar larvae were fed on wild-type (WT) or transgenic Arabidopsis leaves for 2 days, then transferred to diet containing gossypol (1 mg/g) for another day. Data are shown as mean ± SD. * p < 0.05
Fig. 4
Fig. 4
Expression of DpCPV genes in bollworm midgut. The 3rd instar larvae were fed with artificial diet supplemented with E. coli cells expressing indicated fusion proteins for 2 days, and then infected with DpCPV. After 2 days, total RNAs from midgut were extracted and subject to qRT-PCR to detect the expression of DpCPV genes S1 (AY163247) (a), S3 (AY167578) (b) and S4 (AF542082) (c). Expression level of DpCPV genes in the larvae of the GFP group was set to 1. qRT-PCR analysis was biologically repeated for at least three times, error bars represent SD
Fig. 5
Fig. 5
The effect of cysteine protease on plant-mediated bollworm RNAi. The 3rd instar larvae pre-fed with different diets for 2 days were transferred to wild-type (WT) or 35S:dsCYP6AE14 Arabidopsis leaves for 16 h, CYP6AE14 transcript level in midgut was analyzed by qRT-PCR. Larvae were pre-fed with artificial diet supplemented with E. coli cells expressing His-tag fusion proteins of GFP, GhCP1 and AtCP2, respectively (a), or with purified fusion proteins (b), for 2 days; Larvae were pre-fed with WT, 35S:GhCP1(c) or 35S:AtCP2 (d) Arabidopsis leaves, respectively, for 2 days. qRT-PCR analysis was biologically repeated for at least three times, error bars represent SD
Fig. 6
Fig. 6
Effects of cotton overexpressing GhCP1 on bollworm larvae. a Expression of GhCP1 in cotton. Nine 35S:GhCP1 lines were analyzed by qRT-PCR, GhCP1 expression in wild-type cotton (R15) leaves was set to 1; Accumulation of gossypol in midgut and weight increase of tested larvae. 3rd instar larvae were fed on leaves of wild-type (R15) and 35S:GhCP1 (two independent lines: #3 and #4) cotton for 7 days. Gossypol accumulation in midgut (b) and bollworm weight increase (c) were recorded. * P < 0.05, ** P < 0.01; d The level of gossypol equivalents in tested cotton plant leaves; e Expression of DpCPV genes S1 (AY163247), S3 (AY167578) and S4 (AF542082) in bollworm midgut. 3rd instar larvae were fed with wild-type (R15) or 35S:GhCP1#3-1 cotton leaves for one day and then infected with DpCPV; after 2 days, total RNAs from midguts were extracted and subject to qRT-PCR. The expression of DpCPV genes in the larvae pre-fed with wild-type leaves was set to 1. qRT-PCR analysis was biologically repeated for at least three times, error bars represent SD
Fig. 7
Fig. 7
Enhanced bollworm resistance of cotton co-overexpressing GhCP1 and dsCYP6AE14. a Expression level of CYP6AE14. 3rd instar larvae were fed with the indicated leaves (1: wild-type (R15), 2: dsCYP6AE14, 3: 35S:GhCP1 and 4: 35S:GhCP1 dsCYP6AE14) for 16 h. CYP6AE14 expression in midgut of the larvae fed with wild-type (R15) leaves were set to 1; b Accumulation of CYP6AE14 protein in midgut. 3rd instar larvae were fed with wild-type (R15), dsCYP6AE14, 35S:GhCP1 and 35S:GhCP1 dsCYP6AE14 cotton leaves for 2 days. Midgut proteins were detected with an antiserum against CYP6AE14; c Weight increase of the 2nd instar larvae fed with the indicated cotton leaves for 5 and 8 days. * p < 0.05; ** p < 0.01; *** p < 0.001; d The 2nd instar larvae fed with the indicated cotton leaves for 5 days; e The 2nd instar larvae previously fed with the indicated cotton leaves for 5 days were transferred to fresh leaves for another day, then the leaf damage after 1 day of feeding by bollworms was shown (left); gossypol equivalents in cotton leaves were displayed in the right. Error bars represent SD
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